1. Field of the Invention
The present invention relates to sequences of protein L which bind to light chains of immunoglobulins. The invention also relates to hybrid proteins of protein L having the ability to bind to light chains of all Ig and also to bind to light and heavy chains of immunoglobulin G, DNA-sequences which code for the proteins vectors that contain such DNA-sequences, host cells transformed by the vectors, methods for preparing the proteins, reagent apparatus for separating and identifying immunoglobulins, compositions and pharmaceutical compositions which contain the proteins.
2. Description of the Related Art
The invention relates in particular to the DNA-sequence and to the amino acid sequence of the light-chain forming domains of protein L.
Proteins which bind to the constant domains (of high affinity) of the immunoglobulins (Ig) are known. Thus, protein A (from Staphylococcus aureus) (Forsgren, A. and Sjxc3x6quist, J. (1966) Protein A from staphylococcus aureus. I. Pseudo-immune reaction with human gamma-globulin. J. Immunol. 97: 822-827) binds to IgG from various mammal species. The binding of protein A to IgG is mediated essentially via surfaces in the Fc-fragment of the heavy chain of the IgG-molecule, although a certain bond is also effected with surfaces in the Fab-fragment of the IgG. Protein A lacks the ability of binding to human IgG3 and neither will it bind to IgG from several other animal species, such as important laboratory animals, for instance rats and goats, which limits the use of protein A.
Protein G (Bjxc3x6{umlaut over ( )}rck, L. and Kronvall, G. (1984) Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J. Immunol. 133: 969-974; Reis, K., Ayoub, E. and Boyle, M. (1984) Streptococcal Fc receptors. I. Isolation and partial characterization of the receptor from a group C streptococcus. J. Immunol. 132: 3091-3097) binds to heavy chains in human IgG and to all four of its subclasses and also to IgG from most mammals, including rats and goats.
Protein H (xc3x85kesson, P., Cooney, J., Kishimoto, F. and Bjxc3x6rck, L. (1990) Protein Hxe2x80x94a novel IgG binding bacterial protein. Molec. Immun. 27: 523-531) binds to the Fc-fragment in IgG from human beings, monkeys and rabbits. However, the bond is weaker than in the case of protein G and A, which may be beneficial when wishing to break the bond with a weak agent, for instance when purifying proteins which are readily denatured with the aid of antibodies.
Protein M (Applicant""s Patent Application PCT/SE 91100447) binds to the Fc-fragment in IgG from humans, monkeys, rabbits, goats, mice and pigs.
Protein L (Bjxc3x6rck, L. (1988) Protein L, a novel bacterial cell wall protein with affinity to Ig L chains. J. Immunol. 140: 1194-1197), which binds to the light chains in immunoglobulins from all of the classes G, A, M, D and E is known (U.S. Pat. No. 4,876,194). The amino acid sequence and the binding domains of this protein, however, have hitherto been unknown.
The aforesaid proteins can be used in the analysis, purification and preparation of antibodies and for diagnostic and biological research.
The elimination of immunoglobulins, with the aid of plasmapheresis, can have a favourable effect on some autoimmune diseases. A broadly binding protein would be an advantage when wishing to eliminate all classes of antibodies in this context.
It has long been known that infectious conditions can be prevented or cured with the introduction of an immune serum, i.e. a serum which is rich in antibodies against the organism concerned or its potentially harmful product. Examples hereof are epidemic jaundice, tetanus, diphtheria, rabies and generalized shingles. Antibodies against a toxic product may also be effective in the case of non-infectious occasioned conditions. Serum produced in animals against different snake venoms is the most common application in this respect. However, the administration of sera or antibody preparations is not totally without risk. Serious immunological reactions can occur in some cases. Singular cases of the transmission of contagious diseases, such as HIV and hepatitis through the agency of these products have also been described. In order to avoid these secondary effects, it has been desirable to produce therapeutic antibodies in test tubes. A large number of novel techniques for the preparation of antibodies in test tubes have been proposed in recent years. Examples of such techniques are hybridom techniques, synthesis of chima-antibodies and the preparation of antibodies in bacteria. These techniques also enable antibodies to be specially designed which can further widen the use of such molecules as therapeutics, for instance in the case of certain tumour-diseases. In the case of some of these novel methods, however, the product totally lacks the Fc-fragment to which all of the described IgG-binding proteins, with the exception of protein L, bind. There is consequently a need of a process for purifying antibodies for therapeutic use, wherein proteins which have a broad binding activity/specificity, can be of value.
It has long been possible to utilize the antibody reaction with its high grade specificity for diagnosing past or, in some cases, ongoing infections with different parasites. This indirect method of indicating infectious agents is called serology and, in many cases, may be the only diagnostic alternative. In certain cases, it can also be of interest to exhibit specific IgE- or IgA-antibodies. When diagnosing with the aid of serology, the antigen is most often fastened to a solid phase, whereafter serum taken from the patient is incubated with the antigen. Antibodies that have been bound from the patient can then be detected in different ways, often with the aid of a secondary antibody (for instance, an antibody which is directed against the light chains of human antibodies) to which an identifiable label has been attached, such as alkaline phosphatase, biotin, radioactive isotopes, fluorescein, etc. In this context, a protein having a broad Ig binding capacity can be used as an alternative to secondary antibodies.
There are a number of non-therapeutic and non-diagnostic reasons for the necessity to bind antibodies. Antibodies are often used in research, both for detection and for purifying the antigen against which they are directed. All techniques which facilitate the purification of antibodies and, in particular, techniques which enable different classes to be purified, are of interest in this context.
Consequently, there is a serious need of a protein which has a broad binding activity/specificity and which binds to several different classes of immunoglobulins from different animal species. At present, there is no known protein which will bind to all immunoglobulin classes. The earlier known proteins A, G, H and M bind only to heavy chains in IgG.
The known protein L (Bjxc3x6rck et al, 1988) binds to the light "khgr"-chains and xcex3-chains in immunoglobulins of all classes, although the bonds are much weaker on the xcexa-chains. Applicant has charted protein L, has determined the amino acid sequence for protein L, has identified the light-chain binding domains on protein L, and has used these to produce hybrid proteins which possess the IgG-Fc-binding domains of protein G. The Applicant is able to show through protein LG that a protein of broader binding activity/specificity can be produced thereby. The aforesaid proteins A, G. H and M bind to the same surfaces, or to very closely lying surfaces on IgG-Fc. The protein L which binds to light chains can thus be combined with any other functionally similar protein which binds to the Fc-fragment of heavy chains. A similar broadening of the Ig-binding activity is achieved with all alternatives.